Candidate Proteins Interacting with Cytoskeleton in Cells from the Basal Airway Epithelium in vitro

Our research addresses the difficult question of how to dissect a process happening in multiple dimensions inside the cell. In order to exploit all the new information obtained by proteomics methods, it is necessary to integrate it into the process of dynamic remodeling of structures. Our research is relevant to this general area of endeavor where it concerns cytoskeleton. The cytoskeleton consists of actin, microtubules, septins, and intermediate filaments and, in most cells, is anchored to an extracellular matrix. Each cell has a unique arrangement of this network and readjusts it from time to time. To investigate the regulation of these reorganizations, we identified interactors from extracts of four cultured lines representing basal cells from the airway epithelium. Methods: After immunoprecipitation with an antibody against keratin 17, samples were processed by liquid chromatography and tandem mass spectrometry. Samples not undergoing antibody-mediated capture were processed in parallel. Results: The main keratins of basal cells, namely Krt14 (type I) and Krt5 (type II), constituted 67% of the total keratin recovered. Several other intermediate filament proteins, nestin, lamin-B1, and prelamin A/C, were present but not enriched upon immunoprecipitation. Although the class of armadillo-repeat proteins was represented by beta-catenin1 and plakoglobin, other desmosome plaque constituents were absent. Large cytolinkers were represented by the spectraplakin, microtubule-actin cross-linking factor (Macf1), which was enriched by immunoprecipitation, and the plakin, plectin, which was not enriched. Subunits of actin filaments and microtubules, along with numerous proteins associated with them, were recovered in both immunoprecipitated samples and those lacking the capture step. Coefficients of determination were computed based on abundance. The actin-associated proteins, alpha-spectrin and brain-specific angiogenesis inhibitor (Baiaip2l), were modestly correlated with keratin abundance but highly correlated with one another and with the keratin-binding protein, annexin A2. This interaction network resembled the pedestal formed by pathogenic Escherichia coli. Microtubule-associated proteins, dynamin 1-like protein and cytoplasmic dynein 1 heavy chain (Dync1h1), were enriched by immunoprecipitation, suggesting association with keratins, whereas kinesin-1 heavy chain and microtubule-associated protein retinitis pigmentosa 1 (EB1), were not enriched. Dync1h1 abundance was negatively correlated with that of all the septins, suggesting resemblance to a known antagonistic septin-dynein 1 relationship on microtubules. Conclusions: The cell lines showed remarkable uniformity with respect to the candidates interacting with cytoskeleton. The alpha-spectrin-Baiap2l network may link actin filaments to keratin precursor particles. A smaller interaction network centered on Dync1h1 was negatively correlated with all spectrin-Baiap2l constituents, suggesting that it and its binding partners are excluded from the pedestal-like domain.

本研究旨在解析细胞内多维度发生的生物学过程这一棘手问题。为充分利用蛋白质组学方法获取的全部全新信息，需将其整合至结构动态重塑的过程中。本研究与聚焦细胞骨架（cytoskeleton）的相关研究领域高度契合。细胞骨架由肌动蛋白丝（actin）、微管（microtubule）、分隔蛋白（septins）以及中间丝（intermediate filaments）组成，且在多数细胞中锚定于细胞外基质（extracellular matrix）。每个细胞的该骨架网络排布均具有独特性，且会适时对其进行调整。为探究此类骨架重塑的调控机制，本研究从四种代表气道上皮基底层细胞的培养细胞系提取物中筛选得到互作蛋白。

实验方法：使用针对角蛋白17（keratin 17）的抗体进行免疫沉淀（immunoprecipitation）后，采用液相色谱-串联质谱法（liquid chromatography and tandem mass spectrometry）对样本进行处理；同时并行处理未经过抗体介导捕获的对照样本。

实验结果：基底层细胞的主要角蛋白为Krt14（I型）与Krt5（II型），二者占回收总角蛋白的67%。其余数种中间丝蛋白，即巢蛋白（nestin）、核纤层蛋白B1（lamin-B1）以及前核纤层蛋白A/C（prelamin A/C）虽被检出，但在免疫沉淀过程中未出现富集。尽管臂杆重复蛋白家族（armadillo-repeat proteins）以β-连环蛋白1（beta-catenin1）和盘状球蛋白（plakoglobin）为代表，但其他桥粒斑成分并未被检出。大型细胞连接蛋白包括两类：经免疫沉淀后出现富集的spectraplakin家族成员微管-肌动蛋白交联因子（microtubule-actin cross-linking factor, Macf1），以及未出现富集的桥粒斑蛋白（plakin）家族成员网蛋白（plectin）。肌动蛋白丝与微管的亚基，以及大量与之相关的蛋白，在免疫沉淀样本与未进行捕获步骤的对照样本中均有检出。基于蛋白丰度计算了决定系数。肌动蛋白相关蛋白α-血影蛋白（alpha-spectrin）与脑特异性血管生成抑制因子（Baiaip2l）与角蛋白丰度仅呈弱相关，但二者之间以及与角蛋白结合蛋白膜联蛋白A2（annexin A2）之间均呈高度相关。该互作网络与致病性大肠杆菌（pathogenic Escherichia coli）形成的黏附基座结构相似。微管相关蛋白（microtubule-associated proteins）中的动力蛋白1类似蛋白（dynamin 1-like protein）与细胞质动力蛋白1重链（cytoplasmic dynein 1 heavy chain, Dync1h1）经免疫沉淀后出现富集，提示其与角蛋白存在结合；而驱动蛋白1重链（kinesin-1 heavy chain）以及微管相关蛋白视网膜色素变性1（microtubule-associated protein retinitis pigmentosa 1, EB1）则未出现富集。Dync1h1的丰度与所有分隔蛋白的丰度均呈负相关，这与已知的微管上分隔蛋白与动力蛋白1之间的拮抗关系一致。

结论：四种细胞系在与细胞骨架互作的候选蛋白方面表现出显著的一致性。α-血影蛋白-Baiap2l互作网络可能将肌动蛋白丝与角蛋白前体颗粒连接起来。以Dync1h1为核心的小型互作网络与所有α-血影蛋白-Baiap2l网络成员均呈负相关，提示该网络及其结合伴侣被排除在类似黏附基座的结构域之外。